Conferring resistance of plants to either their natural enemies or to those compounds used to protect them from their natural enemies has been accomplished through the techniques of genetic engineering. U.S. Pat. No. 5,306,863 teaches a method of conferring pest resistance by creating genetically transformed plants through the use of trypsin inhibitors derived from legume plants. Similarly, U.S. Pat. No. 5,317,096 describes isolated toxin genes derived from the bacterium Bacillus thuringiensis which may be used to provide transformed plants with resistance to certain insects.
With respect to herbicide resistance, U.S. Pat. No. 5,290,926 teaches the isolation and purification of the plant histidinol dehydrogenase, as well as assays to detect inhibitors of histidinol dehydrogenase (e.g., herbicides) and mutant forms of the wild-type enzyme not inhibited by inhibitors. Transgenic crop plants may thus be produced, containing the non-inhibited mutant enzyme, and these crops may be treated with histidinol dehydrogenase inhibitors to treat weeds.
U.S. Pat. No. 5,145,777 teaches conferring resistance to herbicidal glutamine synthetase inhibitors by amplification of the glutamine synthetase gene by linking it to a sequence which is capable of increasing the expression of the gene. Similarly, U.S. Pat. No. 5,276,268 describes conferring resistance to phosphinothricin, a potent glutamine synthetase inhibitor used in herbicides, through the isolation of phosphinothricin resistance genes and processes of producing plants which are transformed with such genes. U.S. Pat. No. 5,212,296 teaches isolated DNA sequences encoding herbicide-metabolizing cytochrome P450 enzymes and iron-sulfur proteins which assist these enzymes by donating electrons. Both the enzymes and helper proteins may be utilized to transform plants into having the ability to break down herbicides, thus conferring herbicide resistance.
Herbicide safeners, also known as antidodes, are used to protect plants against injury from herbicides. Widely used herbicide safeners include the dichloroacetamides such as dichlormid (R-25788), which are used in combination with thiocarbamate herbicides (e.g., EPTC) and chloroacetanilide herbicides (e.g., metolachlor), on maize and sorghum (FIG. 1). The mode of action of safeners is associated with their ability to stimulate glutathione (GSH)-mediated detoxification of herbicides (Lay, M.-M. et al., Science 189:287-289 (1975)), although the mechanism by which they induce this is unclear.
Etiolated maize seedlings have high-affinity binding activity for the dichloroacetamide safener R-29148 ((R,S)-3-dichloroacetyl-2,2,5-trimethyloxazolidine). Walton, J. D. and Casida, J. E., Plant Physiol. 109:213-219 (1995). R-29148 (Saf) is a commercially used dichloroacetamide safener and is shown in FIG. 1. The characterization of a soluble, high affinity (K.sub.d =120 nM) safener binding activity (SafBA) for [.sup.3 H]Saf in extracts of etiolated maize seedlings has been reported. Walton, J. D. and Casida, J. E., Plant Physiol. 109:213-219 (1995). SafBA is sensitive to boiling and protease treatment. Although present in all tissues of the etiolated seedling, SafBA is most abundant in the coleoptile. There is good correlation between inhibition of binding and safener action among a series of Saf analogs, with dichlormid being the strongest binding antagonist of any compound tested (IC.sub.50 10 nM, IC.sub.50 =inhibitor concentration that reduces specific binding by 50%).
It would thus be desirable to provide the gene responsible for SafBA. It would also be desirable to isolate and purify the gene product. It would also be desirable to control SafBA in plants. It would further be desirable to control SafBA in plants by controlling the expression of the gene encoding the protein responsible for SafBA. It would also be desirable to transform plants using the gene in order to alter their sensitivity to herbicides. It would also be desirable to acquire long-term control over SafBA in plants to provide safener responsiveness in plants which are not safener responsive, and increase safener responsiveness in plants which are safener responsive. It would additionally be desirable to provide an in vitro assay to screen for new herbicides and safeners.